Pannexins form oligomeric membrane channels with a large conductance and physical pore size allowing the exchange of small molecules between the cytoplasm and the extracellular space. One of the three pannexins (Panx1) forms an ATP release channel in several cell types including erythrocytes, airway epithelial cells, immune cells and astrocytes. Cells release ATP through Panx1 channels in response to stimuli such as mechanical stress, inflammation or injury and then ATP serves as a signal propagated to neighboring cells extending the stimulus response. While the evidence for this ATP paracrine release function of Panx1 channels is well-documented in the literature having been gathered independently by many laboratories, most notably by the Dahl laboratory and collaborators, some recent publications have shown that under certain experimental conditions Panx1 channels exhitibed a ten fold smaller unitary conductance then previously reported and with no ATP permeability. We now have discovered that Panx1 can form two channels with distinct permeabilities and conductances within the same cellular environment. Which type of Panx1 channel properties (large conductance and ATP releasing vs. small conductance and small ion releasing) prevails strictly depends on the specific stimulus used for activation. With exclusive voltage activation, the small channel without ATP permeability was observed. Stimulation of the same cell with extracellular potassium ions as a surrogate for physiological stimuli such as mechanical stress, in contrast, led to the large channel conformation with 500 pS conductance and high ATP permeability. Electron micrographs of Panx1 channels with and without K+ stimulation show large differences in pore size between the two conditions. This project is focused on investigating the mechanisms underlying the differential activation mechanisms of the Panx1 channel. The approach involves an integrated combination of electrophysiological analysis, calorimetric measurements of ion-protein interactions and electron microscopy of the phenomenon. The electrophysiology experiments probe the functional dynamics of these stimuli while with a soluble activator (K+) it is possible to image single Panx1 channels in the open and closed states under controlled conditions. Truncation mutants and alanine replacement mutants of Panx1 will be used to determine how the channel opens under the two stimuli and the contribution of the carboxy terminal amino acids to each activation mechanism. This correlated functional-structural approach will be applied to determine pore lining amino acids in the small and large channel conformations. In total, the data obtained in this correlated function-structure study will highlight differences in channel function and conformation, providing a foundation for obtaining new therapeutics or blockers in disease processes where Panx1 channels serve an important function.